Relative roles of GM1 ganglioside, N-acylneuraminic acids, and α2β1 integrin in mediating rotavirus infection

Abstract

N-acetyl- and N-glycolylneuraminic acids (Sia) and α2β1 integrin are frequently used by rotaviruses as cellular receptors through recognition by virion spike protein VP4. The VP4 subunit VP8*, derived from Wa rotavirus, binds the internal N-acetylneuraminic acid on ganglioside GM1. Wa infection is increased by enhanced internal Sia access following terminal Sia removal from main glycan chains with sialidase. The GM1 ligand cholera toxin B (CTB) reduces Wa infectivity. Here, we found sialidase treatment increased cellular GM1 availability and the infectivity of several other human (including RV-3) and animal rotaviruses, typically rendering them susceptible to methyl α-d-N-acetylneuraminide treatment, but did not alter α2β1 usage. CTB reduced the infectivity of these viruses. Aceramido-GM1 inhibited Wa and RV-3 infectivity in untreated and sialidase-treated cells, and GM1 supplementation increased their infectivity, demonstrating the importance of GM1 for infection. Wa recognition of α2β1 and internal Sia were at least partially independent. Rotavirus usage of GM1 was mapped to VP4 using virus reassortants, and RV-3 VP8* bound aceramido-GM1 by saturation transfer difference nuclear magnetic resonance (STD NMR). Most rotaviruses recognizing terminal Sia did not use GM1, including RRV. RRV VP8* interacted minimally with aceramido-GM1 by STD NMR. Unusually, TFR-41 rotavirus infectivity depended upon terminal Sia and GM1. Competition of CTB, Sia, and/or aceramido-GM1 with cell binding by VP8* from representative rotaviruses showed that rotavirus Sia and GM1 preferences resulted from VP8*-cell binding. Our major finding is that infection by human rotaviruses of commonly occurring VP4 serotypes involves VP8* binding to cell surface GM1 glycan, typically including the internal N-acetylneuraminic acid.

Importance: Rotaviruses, the major cause of severe infantile gastroenteritis, recognize cell surface receptors through virus spike protein VP4. Several animal rotaviruses are known to bind sialic acids at the termini of main carbohydrate chains. Conversely, only a single human rotavirus is known to bind sialic acid. Interestingly, VP4 of this rotavirus bound to sialic acid that forms a branch on the main carbohydrate chain of the GM1 ganglioside. Here, we use several techniques to demonstrate that other human rotaviruses exhibit similar GM1 usage properties. Furthermore, binding by VP4 to cell surface GM1, involving branched sialic acid recognition, is shown to facilitate infection. In contrast, most animal rotaviruses that bind terminal sialic acids did not utilize GM1 for VP4 cell binding or infection. These studies support a significant role for GM1 in mediating host cell invasion by human rotaviruses.

FIG 1

Effects of Neu5Acα2Me and antibody to α2β1 integrin on human rotavirus infection of untreated and sialidase-treated MA104 cells. (A) Chemical structures of monomeric N-acetylneuraminic acid and its corresponding methyl glycoside. (B to E) Rotavirus reacted with 10 mM Neu5Acα2Me or diluent (control) was adsorbed to cells treated with sialidase (0.52 U/ml) and/or α2β1 antibody (20 μg/ml). Infected cells were enumerated at 16 h postinfection following indirect immunofluorescent staining. Infectious virus titers are expressed as a percentage of the titers produced in control untreated cells. These control infectious titers, expressed as mean FCFU/ml ± SD, were 7.7 × 10³ ± 1.3 × 10³ (B), 1.2 × 10⁴ ± 0.1 × 10⁴ (C), 8.8 × 10³ ± 0.7 × 10³ (D), and 1.6 × 10⁴ ± 0.3 × 10⁴ (E). Positive-control neutralizing monoclonal antibodies (Ab) were 1A10 (Wa), RV-5:2 (RV-5), and RV-3:1 (RV-3; S12/85). Cells treated with isotype control MOPC21 at 20 μg/ml produced virus titers that were indistinguishable from those of untreated cells.

FIG 2

Effects of Neu5Acα2Me, antibody to α2β1 integrin, and Neu5Gcα2Me on animal rotavirus infection of untreated and sialidase-treated MA104 cells. (A) Chemical structures of monomeric N-acylneuraminic acids and their corresponding methyl glycosides. (B to F) Rotavirus reacted with 10 mM Neu5Acα2Me, 10 mM Neu5Gcα2Me, or diluent (control) was adsorbed to cells treated with sialidase (0.52 U/ml) and/or α2β1 antibody (20 μg/ml). Infected cells were enumerated at 16 h postinfection following indirect immunofluorescent staining. Rotavirus infectious titers are expressed as a percentage of the titers produced in control untreated cells. These control infectious titers, expressed as mean FCFU/ml ± SD, were 1.2 × 10⁴ ± 0.2 × 10⁴ (B), 9.0 × 10³ ± 0.5 × 10³ (C), 1.1 × 10⁴ ± 0.1 × 10⁴ (D), 2.6 × 10⁴ ± 0.1 × 10⁴ (E), and 1.2 × 10⁴ ± 0.2 × 10⁴ (F). Positive control neutralizing monoclonal antibodies (Ab) were 2G4 (RRV), RV-3:1 (CRW-8) and H7/D2 (UK). No positive-control antibody was available for TFR-41 or NCDV. Cells treated with isotype control antibody MOPC21 produced virus titers that were indistinguishable from the titers in untreated cells.

FIG 3

Effects of CTB, a-GM1 treatment, and GM1 or GM3 supplementation on rotavirus infectivity in MA104 cells and mapping UK rotavirus dependence on GM1 to VP4. (A) CTB treatment inhibited infection by human rotaviruses RV-5, RV-3, S12/85, and Wa. (B) CTB inhibition of the infectivity of NCDV, TFR-41, UK, and RRV rotaviruses and reassortant rotaviruses 12-1 and 28-1. Heat-inactivated CTB (Inact.) was included as a negative control, as described before (31). (C) Effects of exposure to 10 mM a-GM1 or a-GM3 on the infectivity of Wa and RV-3 in untreated and sialidase-treated cells. (D) Effects of cell supplementation with 3 μM GM1 or GM3 on Wa and RV-3 infectivity. Rotavirus infectious titers are expressed as a percentage of the titers produced in control untreated cells. These control infectious titers (mean FCFU/ml ± SD) were 3.0 × 10⁴ ± 0.1 × 10⁴ (RV-5), 2.0 × 10⁴ ± 0.1 × 10⁴ (RV-3), 1.8 × 10⁴ ± 0.3 × 10⁴ (S12/85), 2.4 × 10⁴ ± 0.1 × 10⁴ (Wa), 2.6 × 10⁴ ± 0.1 × 10⁴ (NCDV), 2.6 × 10⁴ ± 0.2 × 10⁴ (TFR-41), 1.1 × 10⁴ ± 0.1 × 10⁴ (UK), 1.2 × 10⁴ ± 0.2 × 10⁴ (RRV), 2.7 × 10⁴ ± 0.1 × 10⁴ (12-1), and 7.9 × 10³ ± 0.8 × 10³ (28-1).

FIG 4

NMR spectra of a-GM1. (A) Control ¹H NMR spectrum of a-GM1. STD NMR spectra of a-GM1 in the presence of RRV GST-VP8* (B), Wa GST-VP8* (C), RV-3 VP8* (D), and with GST alone (E) are shown. In all panels, the signals of the two N-acetamido groups' methyl protons at approximately 1.70 ppm are depicted separately on the right at a uniform magnification of ×2.5. All spectra were acquired in deuterated phosphate buffer at 600 MHz and 280 K. (F) Structure of a-GM1 with the binding epitopes of VP8* encircled. The Wa and RV-3 VP8* epitopes include both the Neu5Ac and GalNAc residues (left), whereas the RRV VP8* epitope encompasses only the Neu5Ac moiety (right).

FIG 5

Studies of Sia competition and sialidase sensitivity of cell binding by animal rotavirus VP8*. Cell binding by recombinant VP8* of RRV (A) and NCDV (B) at concentrations (μg/ml) of 5, 50, and 150 to cells placed into suspension using trypsin-EDTA treatment. NCDV GST-VP8* also was tested at 300 μg/ml. The histograms for NCDV VP8* at 150 μg/ml and 50 μg/ml fell close to and between those for 300 μg/ml and 5 μg/ml and are not shown for the sake of clarity. Cell binding by NCDV VP8* at 600 μg/ml (C and D) and CRW-8 VP8* at concentrations (μg/ml) of 5, 50, 150 and 300 (E) also are illustrated. Reduced cell binding by NCDV VP8* (C and D) at 600 μg/ml and CRW-8 VP8* at 5 μg/ml (F and G) following Neu5Acα2Me (C and F) and Neu5Gcα2Me (D and G) treatment is shown. (H) Cellular sialidase treatment reduced binding by NCDV VP8* (600 μg/ml). Reductions in GST-VP8* binding in panels C to G were analyzed for Neu5Acα2Me and Neu5Gcα2Me at concentrations (mM) of 1.25, 2.5, 5.0, and 10. Histograms for NCDV VP8* treated with 1.25 mM Neu5Gcα2Me were indistinguishable from those for untreated NCDV. Data for CRW-8 VP8* incubated with 2.5 mM Neu5Acα2Me or 2.5 mM Neu5Gcα2Me were indistinguishable from those of CRW-8 reacted with the respective Sia at 1.25 mM.

FIG 6

Flow-cytometric analysis of MA104 cell surface GM1 availability and effect of CTB on cell binding by VP8* of Wa, RV-3, and CRW-8. (A) Trypsin-EDTA treatment reduced cell surface GM1 availability. Histograms indicate the GM1 surface exposure, determined by incubation with FITC-CTB, on cells that were placed into suspension by trypsin-EDTA or scraping and rested for 30 min. Each cell preparation was reacted with unconjugated FITC for the controls, which each showed similar histograms. (B and C) Cell binding by recombinant VP8* of Wa (B) and RV-3 (C). Wa and RV-3 GST-VP8* proteins were tested for binding at concentrations (μg/ml) of 5, 50, 150, and 300 to cells placed into suspension using trypsin-EDTA treatment. Effect of cellular treatment with CTB or inactivated (inact.) CTB at 1 μg/ml on cell binding by VP8* of Wa (D), RV-3 (E), and CRW-8 (F). Wa, RV-3, or CRW-8 GST-VP8* protein was added at 300 μg/ml to cells placed into suspension with trypsin-EDTA. (D to F) The histogram of each VP8* bound to untreated cells was indistinguishable from that of the corresponding VP8* bound to cells treated with inactivated CTB.